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Abstract Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

Abstract Adhesion G Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to effect cell signalling. We report here that ADGRF1 can signal through all major G protein classes and identify the structural basis for a previously reported Gαq preference by cryo-EM. Our structure shows that Gαq preference in ADGRF1 may derive from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helix VII and helix VIII at the site of Gα recruitment. Mutational studies of the interface and of contact residues within the 7TM domain identify residues critical for signalling, and suggest that Gαs signalling is more sensitive to mutation of TA or binding site residues than Gαq. Our work advances the detailed molecular understanding of aGPCR TA activation, identifying features that potentially explain preferential signal modulation.